Bio Molecular Research Laboratories, Research, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
Immunology Unit, Research, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
J Immunol Methods. 2020 May;480:112751. doi: 10.1016/j.jim.2020.112751. Epub 2020 Jan 23.
Intracellular DNA triggers interferon release during the innate immune response. Cyclic GMP-AMP synthase (cGAS) senses intracellular double-stranded DNA not only in response to viral infection but also under autoimmune conditions. Measuring the levels of cyclic GMP-AMP (cGAMP) as a second messenger of cGAS activation is important to elucidate the physiological and pathological roles of cGAS. Therefore, we generated monoclonal antibodies against cGAMP using hybridoma technology to test antibody specificity and establish methods to detect intracellular cGAMP. The resulting cGAMP-specific antibody enabled the development of a time-resolved fluorescence energy transfer assay with a quantifiable range of 0.1 nM to 100 nM cGAMP. Using this assay, we detected cellular and tissue cGAMP. We confirmed that the cGAMP antibody successfully targeted intracellular cGAMP through immunocytochemical analyses. These results demonstrated that the cGAMP antibody is a powerful tool that allows determining cGAS involvement in autoimmunity and disease pathology at the cell and tissue levels.
细胞内 DNA 在先天免疫反应中触发干扰素释放。环鸟苷酸-腺苷酸合酶 (cGAS) 不仅在病毒感染时，而且在自身免疫条件下，都能感知细胞内的双链 DNA。测量环鸟苷酸-AMP (cGAMP) 作为 cGAS 激活的第二信使的水平对于阐明 cGAS 的生理和病理作用非常重要。因此，我们使用杂交瘤技术生成了针对 cGAMP 的单克隆抗体，以测试抗体的特异性并建立检测细胞内 cGAMP 的方法。所得的 cGAMP 特异性抗体使我们能够开发出一种具有可量化范围为 0.1 nM 至 100 nM cGAMP 的时间分辨荧光能量转移测定法。使用该测定法，我们检测了细胞和组织中的 cGAMP。我们通过免疫细胞化学分析证实了 cGAMP 抗体能够成功靶向细胞内的 cGAMP。这些结果表明，cGAMP 抗体是一种强大的工具，可用于确定 cGAS 在自身免疫和疾病病理学中的细胞和组织水平上的作用。
